Field of the Invention
The present invention relates to a device and method for bilirubin photo-isomerization to reduce laboratory test interference in mammalian fluids.
Background of the Invention
Bilirubin has spectral and chemical properties that lead to interference with the measurement of common clinical laboratory analytes in patient samples. For example, bilirubin is known to interfere with fluid tests for the measurement of creatinine, cholesterol, and triglycerides. Spectral interference is caused when absorption of a certain wavelength of light is similar between bilirubin and the analyte of interest, thereby artificially increasing the apparent absorption profile of the analyte. Alternatively, bilirubin may absorb or “quench” the signal emanating from an analyte, artificially suppressing the signal. Chemical interference is caused when bilirubin reacts with chemical intermediaries designed to detect an analyte, resulting in compromised measurement of the analyte. These forms of interference may artificially suppress or inflate the values of important diagnostic analytes in a patient sample, reducing the accuracy of clinical chemistry testing. This loss of accuracy may lead serious diagnostic errors and even the administration of inappropriate medical treatments or interventions, which may have unwanted and/or harmful effects on the patient.
Bilirubin is primarily a breakdown byproduct of hemoglobin which is released upon death and decomposition of red blood cells. Jaundice is a clinical condition experienced by many newborn babies as well as children and adults stricken with certain diseases where abnormally high levels of bilirubin have accumulated in their bodies. Its most pronounced symptom is yellow coloring of the skin and eyes.
Normally, bilirubin is conjugated with glucuronic acid in the liver so that it can be solubilized and eliminated from the body through the bile. However, several of the proteins and enzymes that perform this function are not present at the necessary levels in newborns and in certain pediatric and adult diseases. This leads to a rise in bilirubin levels in the blood. Since the bilirubin is not water-soluble it tends to accumulate in body tissues, thereby causing the yellow coloration.
Bilirubin has the chemical structure shown in FIG. 1A. The carboxyl groups form hydrogen bonds with nearby nitrogen atoms, which hides the hydrophilic moieties and increases the molecule's overall hydrophobicity. Bilirubin's hydrophobicity makes it dangerous because it will readily absorb into tissue and cross the blood/brain barrier.
The danger of high levels of bilirubin is that it can be neurotoxic, a condition known as kernicterus. If levels remain elevated there can be irreversible nerve damage, or even death.
Light-mediated isomerization (referred to as phototherapy in therapeutic settings) initiates a structural isomerization of bilirubin to lumirubin, an isomer with substantially different chemical and light absorption properties than bilirubin. Among other differences, the isomerization of bilirubin yields a free carboxyl group that makes lumirubin much more hydrophilic than bilirubin. The magnitude of phototherapy-mediated conversion of bilirubin to lumirubin increases with the intensity of the light, so a device designed to generate light of the proper wavelength and focus that light directly on subject fluid samples will yield substantial benefits in the speed and efficiency of the preparation of fluids for “clean” chemical analyte testing. Since testing labs, in hospitals and outside hospitals, tend to handle scores or hundreds of samples a day, a device maximizing efficiency by reducing time to process bilirubin cleansing will yield a substantial economic benefit to the testing company.
Light-mediated photoisomerization, including phototherapy, is considered to be extremely safe. The only potential risk is damage to the eyes by the intense light. Therefore, it is highly recommended that the eyes of any photoisomerization operators and/or recipients be protected appropriately. Otherwise, no significant side effects of phototherapy have been documented.
The most efficient wavelength for the isomerization of bilirubin is approximately 450 nm, whether applied to the cleansing of fluid samples for testing or the treatment of jaundice. Wavelengths that fall within the range of 400 nm-500 nm, and more specifically 445 nm-475 nm are known to effect isomerization. In addition, a light intensity of at least 6 microWatts per square centimeter per nanometer of light wavelength is needed for phototherapy. This intensity is equivalent to 2.7 milliwatts per square centimeter of 450 nm wavelength light.
Hyperbilirubinemia, an elevation in bilirubin circulating in the blood, can arise from both acute and congenital circumstances. Bilirubin is a natural byproduct of the metabolism of hemoglobin derived from aged or injured red blood cells. Infant hyperbilirubinemia (neonatal jaundice) is common but easily treated by placing the infant under blue lights, known as phototherapy. Unmanaged hyperbilirubinemia, however, leads to kernicterus (brain damage, ataxia) and early mortality.
Solitary bilirubin is insoluble in the blood and cannot be efficiently excreted in this form. It is therefore conjugated and solubilized by the enzyme uridine diphosphate glycosyltransferase 1-A1 (UGT1A1), allowing excretion through the feces and urine. Unconjugated hyperbilirubinemia is a hallmark of Crigler-Najjar Syndrome (CNS) Type 1, a genetic deficiency of UGT1A1. The current standard treatment for CNS is phototherapy, which is conducted within a bed or chamber fitted with blue lights (wavelength of approximately 450 to 530 nm). Light of this wavelength initiates a photo-isomerization reaction which converts unconjugated bilirubin into an isomer known as lumirubin, which is water soluble and readily excreted. However, the onset of puberty is characterized by thickening and pigmentation of the skin, accompanied by a decrease in the body's surface area:volume ratio. These changes represent barriers to light penetration into circulating blood (which contains the majority of bilirubin). Therefore, the efficacy of phototherapy decreases substantially following puberty. Consequently, older CNS patients are particularly susceptible to elevated bilirubin in the blood, which may interfere with clinical chemistry measurements taken on patient samples.
As mentioned above, bilirubin is a byproduct of the natural turnover and destruction of red blood cells, which releases hemoglobin into the blood. Breakdown of hemoglobin releases a heme group which is further catabolized into bilirubin. At physiological blood pH (around pH 7.4), bilirubin's hydrophilic domain is masked by hydrogen bonding, reducing its solubility. However, bilirubin is conjugated to glucuronic acid in the endoplasmic reticulum of hepatocytes by the enzyme uridine diphosphate glycosyltransferase 1-A1 (UGT1A1), disrupting the hydrogen bonds. The resulting configurational and structural changes facilitate bilirubin solubilization and excretion through the liver, kidneys, and intestines. Crigler-Najjar Syndrome (CNS) Type 1 patients lack UGT1A1 activity due to a genetic abnormality and suffer from chronic unconjugated hyperbilirubinemia. While the normal level of unconjugated bilirubin in the blood is 0.2-0.9 mg/dL, CNS patients have ≧20 mg/dL of bilirubin in their blood.
It should be noted that disorders similar to CNS also present in the veterinary context, and that the device and methods described herein apply to all blood samples from mammals, whether human or animal. The same benefits regarding fluid testing protocols for human patients described herein will also apply in the veterinary context.
What is needed is a device and method for processing high-bilirubin fluid samples being tested for other chemical analytes for diagnostic purposes in order to reduce or remove the bilirubin from such samples and thereby minimize the bilirubin's interference with the measurement of such other chemical analytes.